Characterizing Plant Polysaccharides Using Size-Exclusion Chromatography

Leena Pitkänen of Aalto University, based in Aalto, Finland, recently looked over the potential size-exclusion chromatography (SEC) and asymmetric flow field-flow fractionation (AF4) have in separating and characterizing plant polysaccharides. Their findings were published in the Journal of Chromatography A (1).

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Plant polysaccharides are renewable biomacromolecules that are made of monosaccharide units linked with glycosidic bonds. Cellulose is the most abundant plant polysaccharide, followed by different hemicelluloses, such as xylans, mannans, and glucans. These substances are structural components in plants, and while cellulose has been used in many industrial applications, such as textiles, packaging, and food preservation, hemicelluloses are less utilized (2). As green chemistry becomes more prominent, according to scientists, polysaccharides will be used even more as an alternative to fossil fuel-based macromolecules.

Most plant polysaccharides are heterogeneous in their structure, and determining chemical structures is important, as a polysaccharide’s primary structure can define most of its properties, such as solubility and ability to form macrostructures (self-assembly or interactions with other types of molecules). Molar mass distribution (MMD) can also affect the end-use properties of a polysaccharide. To obtain MMD information, separation techniques, such as size-exclusion chromatography (SEC) or field-flow fractionation (FFF), are needed.

SEC, also known as gel permeation chromatography, is a technique where a column with porous beads is used to retain particles smaller than the pore size while rapidly eluting particles larger than the pore (3). Meanwhile, FFF is an online fractionation technique that enables the separation of macromolecules, colloids, nano- and microparticles according to size, chemical composition, or density with excellent resolution over a size range from a few nanometers up to several microns (4). These techniques are commonly coupled online to various detection techniques, and depending on what detectors are involved, other parameters, such as a polysaccharide’s branching, size, and conformation, can also be obtained. Asymmetric flow field-flow fractionation (AF4) has also been used, as it is a “multi-flow” technique that can require more method optimization compared to SEC.

With this review, the scientists summarized the SEC and AF4 methodology that is used for separating and characterizing plant polysaccharides with either industrial or nutritional importance. Both techniques are widely used for separating and characterizing polysaccharides with structural variety, and due to the diverse nature of polysaccharides, separation based on size is important for obtaining molar mass, size, and conformation information over the separated range of macromolecules. Batch measurements, such as static and dynamic light scattering experiments, cannot provide detailed information on the distributions or ranges of molecules. Most plant polysaccharides can be separated using either SEC or AF4, but the latter approach is claimed by Pitkänen to provide a broader separation range, thus serving as a more optimal method for large-sized polysaccharides, samples with colloidal features, polysaccharide aggregates, and rod-like particles. Most AF4 applications are aqueous, meaning that more traditional SEC is still available for polysaccharides and their derivatives since they can dissolve in organic solvents. In the future, due to the push for more environmentally friendly practices, detailed biomacromolecule characterization, such as plant polysaccharides with complex structures, will have even more emphasis.

References

(1) Pitkänen, L. Potential of Size-Exclusion Chromatography and Asymmetric Flow Field-Flow Fractionation in Separation and Characterization of Plant Polysaccharides. J. Chromatogr. A 2025, 1748, 465862. DOI: 10.1016/j.chroma.2025.465862

(2) Bourakadi, K. E.; Semlali, F-Z.; Hammi, M.; Achaby, M. E. A Review on Natural Cellulose Fiber Applications: Empowering Industry with Sustainable Solutions. Int. J. Biol. Macromol. 2025, 1748, 465862. DOI: 10.1016/j.chroma.2025.465862

(3) Size Exclusion Chromatography. ScienceDirect 2021. https://www.sciencedirect.com/topics/materials-science/size-exclusion-chromatography (accessed 2025-4-4)

(4) Field Flow Fractionation. ScienceDirect 2015. https://www.sciencedirect.com/topics/materials-science/field-flow-fractionation (accessed 2025-4-4)